With a long-term goal to build a practical physics of biological material, our Section on Molecular Biophysics measures, characterizes and codifies the forces that govern the organization of all kinds of biological molecules. On pleasing occasion, the way we look at molecules gives us ideas about what goes wrong in molecular disease. Our undertaking is strengthened by its strong connection with physical theory. Through a series of measurements and analyses of the different kinds of forces as revealed in vivo, in vitro, and in computation, we are working with DNA/lipid assemblies for gene therapy; DNA assemblies such as are seen in viral capsids and in vitro; polypeptides and polysaccharides in suspension; and lipid/water liquid-crystals. In all these systems, we simultaneously observe the structure of packing as well as measure intermolecular forces or interaction energies.[unreadable] - - -[unreadable] Molecular forces in molecular disease: [unreadable] How does the frustration of one last step in cholesterol synthesis create the horror of deadly Smith-Lemli-Opitz Syndrome (SLOS)? Why would removal of a double bond create a species that frustrates normal vesicular secretion? Using a mouse model of SLOS, Marjorie Gondre-Lewis, Peng Loh, and Denny Porter investigated the mechanism by which cholesterol affects sterol granule biogenesis in vivo. There, an absence of one of the last two enzymes in the cholesterol biosynthetic pathway results in an accumulation of precursors. These cholesterol-deficient mice show a decrease in the numbers of secretory granules in pancreas, pituitary, and adrenal glands as well as morphologically aberrant granules in exocrine pancreas. Remarkably, regulated secretion could be restored with exogenous normal cholesterol. The possibility of reversal with simple addition of normal cholesterol suggests immediately that physical forces are at work, forces that can differ with small differences in sterols. Thus motivated, Horia Petrache and Daniel Harries in our Lab showed that modification of sterol chemical structure significantly alters membrane physical properties. By X-ray diffraction and osmotic stress, we measured changes in the bending rigidity of bilayers containing either cholesterol or one of its metabolic precursors. Membrane elasticity differs dramatically between slightly different, metabolic-neighbor sterols and varies in the sequence lanosterol < 7-dehydrocholesterol < lathosterol < cholesterol. We interpreted the results in terms of sterol location within lipid structures and modification of lateral stress, a structural feature relevant to interactions within biological membranes. We find that cholesterol is most efficient in enhancing membrane rigidity, a possible clue to why depletion or replacement with other sterols can affect cellular structures. The stiffness of a granule is likely an important factor in the deformations it must endure to undergo secretion. We can see a clear physical logic how physical, mechanical properties conferred by sterols couple with biological action. [unreadable] [unreadable] [unreadable] Van der Waals forces: [unreadable] The dominant force that coheres membranes and proteins, source of the powerful surface tension at membrane interfaces, van der Waals forces are again the dominant -- perhaps sole -- attraction that creates membrane multilayers or allows membranes to adhere to artificial surfaces. The key has been to begin with the elements of physical theory that relate the polarizability of materials to the fluctuations of charges within them. From this we have been able to design experiments that show how macromolecular organization responds to deliberate changes in solution properties. Progress is thus through a tight coupling of modern electromagnetic theory of structured materials coupled with experiments and measurements that reveal electromagnetic properties. [unreadable] In the present instance, we have teamed with groups that measure absorption spectra in order to formulate and to compute charge fluctuation forces involving lipids, water, and ions, as well as synthetic structures such as carbon nanotubes. The results have shown how charge fluctuation forces conferred by ions in solution can modify forces between lipid membranes. We have measured those forces as well as computed van der Waals charge fluctuation forces in those same systems. [unreadable] The differences between different kinds of ions show clearly how the identity of different salts will impact forces that organize large structures. More to the interactions of membranes, because ions vary widely in their effects on biological materials, ion "specificity" beyond simple charge properties is a major issue in biology. One overlooked property of ions is the polarizability, the ability of the charge to shift or fluctuate, a property seen in charge fluctuation forces. Another surprising feature of ions is the tendency to stick to charged bilayers, stick to an extent beyond what is expected from charge-charge attraction. This stickiness changes the way membranes interact, it also introduces strains that can alter the way proteins are accommodated and are able to change conformation as in the opening and closing of trans-membrane ionic channels.[unreadable] Further, we have seen how the attraction between membranes varies when salts of chloride vs. bromide are dissolved in the intervening water. Membrane multilayers will swell by 50% with bromide but not with chloride. We have reformulated van der Waals forces between membranes to show how they would respond to changes in solutions so as to have a strategy to control membrane assembly. One unexpected by-product has been collaboration with engineers using our equations to design production procedures for thin-film resistors in computer chips. We expect the collaboration to work to our benefit by providing us with experimental data that are used to compute van der Waals forces. [unreadable] We also progressed in extending the Lifshitz theory of van der Waals interactions in stratified media like lipid multilamellar systems to be able to compute forces between bodies with extended interfaces. These can range from the practical ? the composite media of electric insulators ? to the biological ? the action of extended polymer layers on biological membranes. [unreadable] [unreadable] Solute control of molecular association: [unreadable] To monitor how small adherent molecules affect molecular association, we measured the changes of binding free energy vs. change in water activity for the specific binding of cyclodextrin with an adamantane derivative. The dependence of the binding constant on osmotic pressure, using different salts and neutral agents, suggests a release of 15-25 water molecules from the interacting surfaces upon association, depending on the type of solute used. The observed dependence of binding free energy and enthalpy with added solute indicates that these osmolytes are interacting primarily enthalpically with these surfaces. [unreadable] A remarkable number of cellular processes are controlled by the osmotic action of small solutes. These include gating of ionic channels and specific versus non-specific DNA?protein interactions regulating gene expression. Osmotic sensing at the molecular level can probe the forces acting between and within macromolecules. By varying the salt or neutral ?osmolyte? concentration in the bathing solution, we control osmotic pressure. We measure the effect of the varied pressure on the association of carbohydrates with membrane protein channels. A single event of ?-cyclodextrin (CD) nesting in the lumen of a maltoporin channel is seen as a transient drop in ionic current due to the partial occlusion of the channel pore. The change in equilibrium constant of CD binding to the channel vs. solution osmotic pressure translates into the number of water molecules released in the specific binding.